A field-deployable colorimetric bioassay for the rapid and specific detection of ribosomal RNA.

Rapid and specific on-site detection of disease-causing or toxin-producing organisms is essential to public health and safety. Many molecular recognition methods target ribosomal RNA sequences due to their specificity and abundance in the cell. In this work RNA targets were identified and quantified using a colorimetric bioassay. Peptide nucleic acid (PNA) probes were used to capture RNA targets, and a micrococcal nuclease digestion was performed to remove all non-target nucleic acids, including single base mismatches flanked by adenines or uracils. Perfectly-matched PNA-RNA hybrids remained intact and were detected using the symmetrical cyanine dye 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Assay applicability to complex samples was demonstrated using mixtures containing RNA sequences from two related, harmful algal bloom-causing Alexandrium species. Target RNA was detected even in mixtures with mismatched sequences in excess of the perfect match. The fieldability of the assay was tested with a portable two-wavelength colorimeter developed to quantify the dye-indicated hybridization signal. The colorimeter sensing performance was shown to be comparable to a laboratory spectrophotometer. This quick, inexpensive and robust system has the potential to replace laborious identification schemes in field environments.

[1]  B. Nordén,et al.  Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. , 1997, Biochemistry.

[2]  David Yaron,et al.  A structural model for cyanine dyes templated into the minor groove of DNA , 2006 .

[3]  G. Felsenfeld,et al.  MICROCOCCAL NUCLEASE AS A PROBE OF DNA CONFORMATION. , 1964, Biochemistry.

[4]  Miaomiao Wang,et al.  Spontaneous Assembly of Helical Cyanine Dye Aggregates on DNA Nanotemplates , 1999 .

[5]  R. Gil,et al.  1H NMR and optical spectroscopic investigation of the sequence-dependent dimerization of a symmetrical cyanine dye in the DNA minor groove. , 2005, Biochemistry.

[6]  H. Drew.  Structural specificities of five commonly used DNA nucleases. , 1984, Journal of molecular biology.

[7]  V V Demidov,et al.  Stability of peptide nucleic acids in human serum and cellular extracts. , 1994, Biochemical pharmacology.

[8]  B. Nordén,et al.  Genetic screening using the colour change of a PNA-DNA hybrid-binding cyanine dye. , 2002, Nucleic acids research.

[9]  D. Anderson,et al.  Identification and enumeration of Alexandrium spp. from the Gulf of Maine using molecular probes , 2005 .

[10]  B. Armitage,et al.  Molecular Recognition of PNA-Containing Hybrids: Spontaneous Assembly of Helical Cyanine Dye Aggregates on PNA Templates , 1999 .

[11]  E. Berdalet,et al.  DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION , 1999 .

[12]  R A Laskey,et al.  High sequence specificity of micrococcal nuclease. , 1981, Nucleic acids research.

[13]  M. Komiyama,et al.  Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system. , 2004, Nucleic acids research.

[14]  R. Marchelli,et al.  Fast and easy colorimetric tests for single mismatch recognition by PNA-DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-beta-cyclodextrin. , 2007, Journal of biochemical and biophysical methods.

[15]  Van Dolah Fm.  Marine algal toxins: origins, health effects, and their increased occurrence. , 2000 .

[16]  W. Litaker,et al.  Enhanced detection levels in a semi-automated sandwich hybridisation assay using a peptide nucleic acid (PNA) signal probe , 2006 .

[17]  H. Aburatani,et al.  PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection. , 2003, Journal of the American Chemical Society.

[18]  D. Tautz,et al.  Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted , 2006, Nucleic Acids Research.

[19]  Peter E. Nielsen,et al.  PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules , 1993, Nature.

[20]  D. J. Rose,et al.  Characterization of antisense binding properties of peptide nucleic acids by capillary gel electrophoresis. , 1993, Analytical chemistry.

[21]  R. Marchelli,et al.  Unconventional method based on circular dichroism to detect peanut DNA in food by means of a PNA probe and a cyanine dye. , 2005, Chirality.

[22]  William W. West,et al.  The Dimeric State of Cyanine Dyes , 1965 .

[23]  P. Nielsen,et al.  PNA-nucleic acid complexes. Structure, stability and dynamics , 1996, Quarterly Reviews of Biophysics (print).

[24]  Combination of peptide nucleic acid beacon and nuclease S1 for clear-cut genotyping of single nucleotide polymorphisms. , 2007, Analytical biochemistry.

[25]  B. Glynn,et al.  Recent advances in the development of nucleic acid diagnostics , 2010, Expert review of medical devices.

[26]  Peter E. Nielsen,et al.  Peptide nucleic acids (PNA) : oligonucleotide analogues with an achiral peptide backbone , 1992 .

[27]  S. Boppart,et al.  Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells , 2003, Applied and Environmental Microbiology.

[28]  J. Coull,et al.  PNA solubility enhancers , 1998 .

[29]  B. Nordén,et al.  Interactions of DNA binding ligands with PNA-DNA hybrids. , 1994, Nucleic acids research.

[30]  B. Armitage,et al.  DNA-templated assembly of helical cyanine dye aggregates: a supramolecular chain polymerization. , 2004, Accounts of chemical research.

[31]  A. Whitney,et al.  Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory. , 2006, Clinica chimica acta; international journal of clinical chemistry.

[32]  W Hörz,et al.  Sequence specific cleavage of DNA by micrococcal nuclease. , 1981, Nucleic acids research.

[33]  H. Stender,et al.  PNA for rapid microbiology. , 2002, Journal of microbiological methods.